A study of the kinetics and mechanism of rabbit muscle <scp>l</scp>-glycerol 3-phosphate dehydrogenase

Bentley, Philip; Dickinson, F M

doi:10.1042/bj1430019

Cited by 23 publications

(25 citation statements)

References 17 publications

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“…The latter was used to calculate k cat = 130 s −1 , and a value of K m = 0.13 mM for the reactive free carbonyl form of DHAP was calculated using (1 -f hyd ) = 0.55 for the fraction of DHAP present in the free carbonyl form (Scheme 3) (17). These data (Table 1) are in agreement with the published kinetic parameters for GPDH (18).…”

Section: Resultssupporting

confidence: 79%

“…The sugar L-glycerol 3-phosphate is a noncompetitive product inhibitor of the GPDH-catalyzed reduction of DHAP when either NADH or DHAP is the varied substrate; but, the inhibition with respect to NADH changes to uncompetitive when the reaction is carried out in the presence of saturating concentrations of DHAP (19). These results provide strong evidence for an ordered mechanism, with NADH binding first followed by DHAP (18,19). A similar ordered mechanism is assumed to hold for reduction of the "two-part substrate" [glycolaldehyde + phosphite] by NADH, with the cofactor binding first, followed by binding of the substrate pieces GLY and phosphite dianion (Scheme 4).…”

Section: Discussionmentioning

confidence: 83%

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A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

2008

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The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD + , GPDH) saturated by NADH is (1.0×10 6 M −1 s −1 )/(8.7×10 −3 M −1 s −1 ) = 1.1×10 8 , which was used to calculate an intrinsic phosphate binding energy of ≤ −11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH (K d > 0.1 M), but the bound dianion strongly activates GLY towards enzymecatalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M −2 s −1 )/(8.7×10 −3 M −1 s −1 ) = 5×10 5 M −1 , which was used to calculate an intrinsic phosphite binding energy of −7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer and phosphoryl transfer reactions. Structural data provides strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring: (1) a binding pocket complementary to the reactive substrate fragment, and which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate; (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates leads to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.Enzymes are protein catalysts that effect much larger rate accelerations than those observed for small molecule catalysts. The larger rate accelerations for enzymes are a consequence of the greater binding affinities of enzymes for their reaction transition states (1). Enzyme catalysis is so efficient that the release of products would be intolerably slow if they were to bind with the same affinity as the transition state. For example, the ca. 30 kcal/mol transition state intrinsic binding energy estimated for the enzymatic decarboxylation of orotidine 5′-monophosphate (OMP) 1 catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) (2) is even larger than the 20 kcal/mol binding energy associated with the effectively irreversible binding of biotin to avidin (3). Consequently, in order to avoid this free energy "trap", enzymes generally bind their substrates/products much more weakly than the reaction transition state.

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Section: Resultssupporting

confidence: 79%

Section: Discussionmentioning

confidence: 83%

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

2008

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“…If ES2 arises principally by dissociation from ES1S2 then this process is likely to be more marked if the rate of conversion into ESS2 is lower. The mechanism shown in Scheme 1 has also been found to describe the oxidation of primary and secondary alcohols by horse liver alcohol dehydrogenase (Dalziel & Dickinson, 1966) and the oxidation of glycerol 3-phosphate by rabbit muscle glycerol 3-phosphate dehydrogenase (Bentley & Dickinson, 1974). In those cases also the formation of the complex ES2 is manifest when the rate of the catalytic step is relatively low.…”

Section: Discussionmentioning

confidence: 88%

A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates

Dickenson¹,

Dickinson²

1975

Biochemical Journal

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The kinetics of ethanol oxidation by NAD+, and acetaldehyde and butyraldehyde reduction by NADH, catalysed by yeast alcohol dehydrogenase, were studied in the pH range 4.9--9.9 at 25 degrees C and in the temperature range 14.8--43.5 degrees C at pH 7.05. The kinetics of reduction of acetaldehyde by [4A-2H]NADH at pH 7.05 and pH 8.9 at 25 degrees C were also studied. The results of the kinetic experiments indicate that the mechanism of catalysis, previously proposed on the basis of studies at pH 7.05 and 25 degrees C (Dickinson & Monger, 1973), applies over the wide range of conditions now tested. Values of some of the initial-rate parameters obtained were used to deduce information about the pH- and temperature-dependence of the specific rates of combination of enzyme and coenzymes and of the dissociation of the enzyme--coenzyme compounds. Primary and secondary plots of initial-rate data are deposited as Supplementary Publication SUP 50043 (20 pages) with the British Library (Lending Division), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies may be obtained under the terms indicated in Biochem. J. (1975) 145, 5.

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“…Velocity constants for the reactions of enzyme and coenzymes are defined in Scheme 1 of Bentley & Dickinson (1974). The second-order velocity constants for the reaction between enzyme and NADH (k+1) were measured in sodium phosphate buffers, over a pH range 6-9 by using the stopped-flow apparatus as a filter fluorimeter.…”

Section: Estimation Ofthe Dissociation Constant Ofthe Enzyme-na Dh Comentioning

confidence: 99%

“…This information is presented here. The results of detailed initial-rate studies together with discussion of possible mechanisms of catalysis are presented in Bentley & Dickinson (1974). A certain amount of information about coenzyme binding to glycerol 3-phosphate dehydrogenase may be found in the literature (Kim & Anderson, 1969;Reynolds, 1971) but usually for conditions of temperature, pH and reaction-medium composition slightly different from those ofour kinetic studies.…”

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confidence: 99%

A study of the coenzyme-binding characteristics of rabbit muscle l-glycerol 3-phosphate dehydrogenase

Bentley

Dickinson

1974

Biochemical Journal

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1. The binding of NAD(+) and NADH to glycerol 3-phosphate dehydrogenase was studied in the pH range 6.0-9.0 at 25 degrees C and in the temperature range 16-43 degrees C at pH7.0. 2. The second-order velocity constants for the combination of NADH with the enzyme in the pH range 6.0-9.0 and for the combination of NAD(+) with the enzyme at pH6.0 were determined. 3. The velocity constant for the dissociation of the enzyme-NAD(+) complex at pH6.0 was measured.

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A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

Cited by 23 publications

References 17 publications

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates

A study of the coenzyme-binding characteristics of rabbit muscle l-glycerol 3-phosphate dehydrogenase

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A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

Cited by 23 publications

References 17 publications

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates

A study of the coenzyme-binding characteristics of rabbit muscle l-glycerol 3-phosphate dehydrogenase

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A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion